Ras proteins are key regulators of growth, differentiation and malignant transformation. In addition, these proteins are implicated in synaptic function and region-specific learning and memory functions in the brain.
As shown schematically in FIG. 1, Ras proteins cycle between inactive GDP-complexed and active GTP-complexed states. GTPase-activating proteins (GAPs) inactivate Ras proteins by stimulating hydrolysis of the bound GTP to GDP, whereas guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating release of GDP and the uptake of GTP. So essential are GEFs to Ras action, that genetic loss of GEF function has similar effects to those induced by loss of the Ras proteins themselves. Loss of GEF function can be circumvented by mutations that constitutively activate the Ras proteins, such as an oncogene mutation, or, in some cases, through loss of GAP activity. Activated Ras proteins, which are localized at the plasma membrane, transmit signals from tyrosine kinases to a cascade of serine/threonine kinases, which delivers the signals to the cell nucleus.
Activation of Ras can result in the activation of the mitogen-activated protein (MAP) kinase (also known as extracellular-signal regulated kinase, or ERK) pathway. For example, a receptor tyrosine kinase is activated by a peptide mitogen such as epidermal growth factor (EGF). The EGF-stimulated receptor undergoes autophosphorylation of specific tyrosine residues in its cytoplasmic domain which creates phosphotyrosyl binding sites for the Src homology 2 (SH2) and/or phosphotyrosyl binding (PTB) domains of certain adapter proteins. The adapter protein becomes autophosphorylated on association with activated receptor tyrosine kinases. The GEF is stably associated with the adapter protein which, upon autophosphorylation, mediates translocation of the GEF to the plasma membrane. The GEF then activates the Ras protein. Activated Ras relays its signal downstream through a cascade of cytoplasmic proteins, including Raf-1 serine/threonine kinase. The Ras:Raf association promotes translocation of the normally cytoplasmic Raf protein to the plasma membrane, where subsequent events lead to the activation of its kinase function. Upon activation, Raf phosphorylates and activates two MAP kinases (also known as MEKs). MEKs directly associate with the catalytic domain of Raf-1 and are phosphorylated by Raf. Activated MEKs function as dual-specificity kinases and phosphorylate tandem threonine and tyrosine residues in the MAP kinases to activate them. Once activated, the MAP kinases translocate to the nucleus where they phosphorylate and activate a variety of substrates.
Rap proteins, members of the Ras small GTPase superfamily, can inhibit Ras signaling of the Ras/Raf-1(a serine/threonine kinase)/MAP kinase pathway or, through B-Raf, can activate MAP kinase. Rap1 consists of two isoforms, Rap1A and Rap1B, which differ mainly at the C-terminus. Characteristic features of Rap1 are its geranylgeranyl modification at the C-terminus, which is responsible for membrane attachments, and a threonine residue at position 61. In most other GTPases, the corresponding residue is a glutamine. Rap proteins, like Ras proteins, cycle between inactive GDP-complexed and active GTP-complexed states. GEFs are required to activate Rap proteins by stimulating the release of GDP and the uptake of GTP.
Constitutive activation of the Ras pathway contributes to malignant transformation. In fact, the Ras gene has been implicated in many human cancers, including lung cancer, breast cancer, colorectal cancer, exocrine pancreatic cancer, and myeloid leukemia. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein since Ras must be localized in the plasma membrane and must bind with GTP in order to transform cells. Gibbs et al., 53 MICROBIOL. REV. 171–286 (1989).
Targeting components of the Ras signaling pathways has been proposed as one approach for the development of anti-Ras drugs for cancer treatment. One potential approach for targeting Ras for cancer treatment involves the use of farnesyltransferase inhibitors (FTIs). Inhibition of farnesyl-protein transferase, and thereby of farnesylation of the Ras protein, blocks the ability of Ras to transform normal cells to cancer cells. Certain inhibitors of Ras farnesylation cause an increase in soluble Ras which can act as a dominant negative inhibitor of Ras function. While soluble Ras in cancer cells can become a dominant negative inhibitor, soluble Ras in normal cells would not be an inhibitor. A cytosol-localized and activated form of Ras acts as a dominant negative Ras inhibitor of membrane-bound Ras function. Gibbs et al., 86 PROC. NAT'L ACAD. SCI. USA 6630–34 (1989). FTIs block Ras function by preventing its post-translational modification by the farnesyl isoprenoid.
Intervention of Ras signaling at multiple or various points can significantly impact the ability of Ras to cause cellular transformation. Since Ras protein function is believed to be crucial to so many cellular processes, targeting only a subset of Ras functions by downstream intervention may provide significant advantages. Thus, there remains a need for identifying additional means for disrupting the Ras pathway. Applicants have discovered four new targets, namely GEFs specific for Rap1A, for disrupting the Ras pathway.